The growing use of polymerase chain reaction (PCR) in molecular diagnostic has made it desirable to develop reagents and methods capable of simple and effective preservation and processing of a large number of biological samples.
Traditional alkaline lysis requires the following steps: concentrating or pelleting cells diluted in growth media; centrifuging or vortexing; lysing cells with alkaline detergent; shaking and/or agitating lysate; adding neutralization buffer; filtering and/or manipulating sample to remove the flocculent mass; adding a solid phase carrier; and adding binding buffer. Additional purification steps are generally required to remove the detergents and salts as follows: addition of solid phase carriers and addition of a binding buffer.
A frequently used source in DNA diagnostic is blood although buccal swabs and tissue biopsies are also often used, especially for the cancer testing. Currently, tissue samples are stored and preserved frozen, formalin-fixed or paraffin-imbedded. The use of formalin-fixed or paraffin-imbedded samples require a multi-step process to render DNA from these samples usable for PCR. Blood storage for DNA diagnostic purposes requires refrigeration, freeze-drying or freezing. Alternatively, blood samples are applied to a card, such as an FTA CARD (Invitrogen) and store dry at room temperature.
Leal-Klevez et al. developed a method for storing blood and tissue samples at −20 C in 20% ethylene glycol-propylene glycol aqueous solution. In this protocol, DNA can be extracted from the stored sample by the proteinase K-phenol extraction procedure.
U.S. Pat. No. 6,602,718 describes methods and reagents for stabilizing RNA and DNA in biological samples using an aqueous solution or dispersion of cationic detergent supplemented with additives including chaotropic salts, at a pH range from about 2 to about 12.
Various methods of sample processing have been used to prepare DNA for the PCR analysis. DNA is typically extracted using phenol-based methods, salt-alcohol precipitation method or adsorption on column methods. A review of these methods is provided in Current Protocols pp. 2.0.1-2.4.5. Also, U.S. Pat. Nos. 5,346,994 and 5,945,515 described reagents and methods for DNA isolation. These reagents and methods provide substantially pure DNA. However, these are time consuming and not easy adaptable for automation. Methods for performing PCR on samples without prior isolation of DNA have been developed such as: heating samples at 94° C. (Mercier et al.), microwave irradiation (Ohhara et al.) and treatment with thermophilic protease (Belly et al.). Many of these methods relay on 40 cycle-PCR amplification. A 40-cycle PCR amplification is prone to artifacts and cannot be reliably used for diagnostic purposes.
Previously, alkaline lysis was used to prepare blood and other samples directly for PCR, without purification of DNA. Rudbeck et al. used 0.2 M NaOH to lyse at room temperature whole blood, semen and epithelial cells. The lysate was neutralized and used directly for PCR 20 mM NaOH was not effective in releasing DNA usable for PCR. Ashen et al. modified this protocol by first isolating blood leukocytes by centrifugation and lysing. The isolated leukocytes were lysed in 0.2 M NaOH at room temperature. The lysate was neutralized and used for PCR.
Truett et al. used 25 mM NaOH-0.2 mM disodium EDTA buffer, pH 12 to lyse mouse tissue samples by heating at 95 C. After heating, sample lysates were neutralized and used for PCR. This method was not very effective since it involved 40 cycles of PCR to amplify DNA fragments. Also, neutralization of the alkaline lysate added one step to the protocol and made strict requirements for accurate determination of pH in the lysate.
Lin et al. described preparation of DNA from blood by first removing hemoglobin from the whole blood by clotting the blood for a minimum 2 h followed by overnight storage and centrifugation. Resulted serum was subjected to lysis in 18 mM KOH at 37 C, neutralized and used for PCR.
Rapid protocols for DNA analysis by PCR were also detailed for bacteria. Sandhu et al. boiled bacteria colonies in water. Joshi et al. used bacteria directly for PCR without boiling. An FTA CARD method for storing and processing of bacterial cells were also reported (Rogers et al.).
There is now a need for a reliable and shelf-stable reagents and methods for the ambient temperature storage and which are compatible with reagents and methods for processing biological samples for direct PCR. Unexpectedly, it has now been found that reagents comprised of glycols preserve DNA and secure long-term ambient temperature storage of biological samples.